1. Field of the Invention
Embodiments presented herein relate to methods and compositions for the fermentative production of ethanol from D-xylose using yeast.
2. Background
Metabolic engineering of microorganisms is often an effective means to produce commercially a number of chemicals that may be used for multiple applications (see, e.g., Lee, S. Y., et al. Macromol. Biosci. 4:157-164 (2004)). One chemical that has garnered much interest is ethanol. Although ethanol has a number of uses, it is most commonly used as fuel. As a fuel, ethanol is a low value product with much of the cost of its production attributed to the cost of raw materials. It would be desirable, therefore, to develop ethanolgens and fermentation processes for the production of ethanol from readily available, inexpensive starting materials. These starting materials may be, for example, lignocellulosics. These lignocellulosics may be derived, for example, from renewable biomass waste streams from food, paper pulping operations, agricultural residues and recycled paper from municipalities.
Lignocellulose is approximately 30% D-xylose (see Ryabova, O. B., et al. “Xylose and Cellobiose Fermentation to Ethanol by the Thermotolerant Methylotrophic Yeast Hansenula polymorpha,” FEMS Yeast Res. 4:157-164 (2003)). Xylose is a “wood sugar” with the IUPAC designation (2S,3R,4S,5R)-oxane-2,3,4,5-tetrol.
Only a relatively small number of wild type microorganisms can ferment D-xylose. These microorganisms are generally not suitable for large-scale fermentation. This unfavorability may arise, for example, as a result of unfamiliarity with the microorganisms, difficulty obtaining the microorganisms, poor productivity and/or growth on pretreated lignocellulosics or unsatisfactory yield when grown on mixed sugars derived from biomass. C. Abbas, “Lignocellulosics to ethanol: meeting ethanol demand in the future,” The Alcohol Textbook, 4th Edition. (K. A. Jacques, T. P. Lyons and D. R. Kelsall, eds). Nottingham University Press, Nottingham, UK, 2003, pp. 41-57.; Cite 2 C. Abbas, “Emerging biorefineries and biotechnological applications of nonconventional yeast: now and in the future,” The Alcohol Textbook, 4th Edition. (K. A. Jacques, T. P. Lyons and D. R. Kelsall, eds). Nottingham University Press, Nottingham, United Kingdom, 2003, pp. 171-191.
Yeasts are considered the most promising microorganisms for alcoholic fermentation of xylose (see Ryabova, supra). They have larger cells than bacteria, are resistant to viral infection, and tend to be more resistant to negative feedback from ethanol. Furthermore, yeast growth and metabolism have been extensively studied for a number of species. A number of yeasts are known to naturally ferment D-xylose. These include Pichia stipitis, Candida shehatae, and Pachysolen tannophilus (see Ryabova, supra; C. Abbas 2003). The common brewer's yeast Saccharomyces cerevisiae is not known to ferment D-xylose naturally, but a number of strains of biologically engineered S. cerevisiae that do ferment D-xylose have been reported.
As shown in FIG. 1, D-Xylose metabolism in yeast is has been reported to proceed along a pathway similar to that of glucose via pentose phosphate pathway. Carbon from D-xylose is processed to ethanol via the glycolytic cycle or to CO2 via respiratory TCA cycle
It has been proposed that one bottleneck involved in D-xylose fermentation is the hydrolysis of xylan, which is the major component of hemicellulose to monosaccharides (see Ryabova, supra). One approach to overcoming this bottleneck is by using “simultaneous saccharification and fermentation” (SSF). This is a process in which pretreated lignocellulose is hydrolyzed by cellulases and hemicellulases while the hexoses and pentoses produced by this hydrolysis (including xylose) are fermented to ethanol. This would allow continuous conversion of the sugars to ethanol, preventing their accumulation in the medium.
A potential drawback of SSF is the difference in the optimal temperature at which cellulases and hemicellulases are active (at least about 50° C.) that are compatible with the optimal temperature for yeast growth and fermentation of xylose (about 30° C.). One solution to this potential drawback is to perform SSF using the thermotolerant methylotrophic yeast Hansenula polymorpha (also known as Pichia angusta). This yeast has been reported to have optimum and maximum growth temperatures of 37° C. and 48° C., respectively. These temperatures are higher than those tolerated by most other ethanol producing yeasts (Ryabova, et al.). Furthermore, Ryabova, et al. reported that under some conditions H. polymorpha is able to naturally ferment D-xylose (see also Voronovsky, A. Y., et al., “Expression of xylA Genes Encoding Xylose Isomerases From Escherichia coli and Streptomyces coelicolor in the Methylotrophic Yeast Hansenula polymorpha” FEMS Yeast Res. 5(11): 1055-62 (2005)). Behavior of H. polymorpha under high temperatures is reported, for instance, in Escalante, J., et al., “Biomass Production by a Thermotolerant Yeast: Hansenula polymorpha” J. Chem. Tech. Biotechnol. 48: 61-70 (1990); Tsiomenko, A. B., et al., “Secretory Heat-Shock Protein of the Thermotolerant Yeast Hansenula polymorpha. Identification and Comparative Characteristics” Biochemistry (Moscow) 62(2): 123-128 (1997); Lindquist, S. & Kim, G., “Heat-shock Protein 104 Expression is Sufficient for Thermotolerance in Yeast” Proc. Natl. Acad. Sci. USA 93: 5301-5306 (1996); Guerra, E., et al. “Hypoxia Abolishes Transience of the Heat-shock Response in the Methylotrophic Yeast Hansenula polymorpha” Microbiology 151: 805-811 (2005).
Therefore it would be advantageous to develop strains of H. polymorpha with an increased ability to produce ethanol from lignocellulosic starting materials, including the C5 sugar, D-xylose. The present teachings may provide these advantages and/or others, and may provide further advantages that one of skill in the art will readily discern from the detailed description that follows.